identifying secondary peaks

[bisnettrj2]

What type of filter are you using (cellulose, PTFE, GHP, PVDF)? Is it a syringe filter? What type of syringe (PTFE/polypropylene? What about the plunger - any rubber on it?)? Have you tried using the same filtration setup to filter A mobile phase and then inject it to see if you're getting an interference from the filters?

[HPLCaddict]

For completeness, these are the chromatographic parameters from EP:
Column:
— size: l = 0.15 m, Ø = 3.9 mm,
— stationary phase: octadecylsilyl silica gel for chromatography R (5 µm).
Mobile phase: acetonitrile R, water R (10:90 V/V).
Flow rate: 1 mL/min.

Don't know where your friend got that procedure from...as Peter already pointed out, 95% ACN in your A solvent doesn't make any sense for a reversed-phase application.

[jackie1016]

What type of filter are you using (cellulose, PTFE, GHP, PVDF)? Is it a syringe filter? What type of syringe (PTFE/polypropylene? What about the plunger - any rubber on it?)? Have you tried using the same filtration setup to filter A mobile phase and then inject it to see if you're getting an interference from the filters?

> im using PVDF filter, polypropylene syringe filter, the plunger is made out of plastic. and im filtering my mobile phase A first using that same filtration set up then i will filter the standards.,

> i have also injected the mobile phase A that i filtered using that filtration set up

the chroms is the one i posted before using 5%ACN MPA and 20% MPB.

[jackie1016]

For completeness, these are the chromatographic parameters from EP:
Column:
— size: l = 0.15 m, Ø = 3.9 mm,
— stationary phase: octadecylsilyl silica gel for chromatography R (5 µm).
Mobile phase: acetonitrile R, water R (10:90 V/V).
Flow rate: 1 mL/min.

Don't know where your friend got that procedure from...as Peter already pointed out, 95% ACN in your A solvent doesn't make any sense for a reversed-phase application.

> yes i have seen this one from the online pharmacopeia, this is monograph for the API only and not the tablets

im still clarifying the procedure with my friend and still waiting for the reply as to where she get that procedure...

il post an update on the procedure after i have confirm with the source of the monograph

[jackie1016]

Thiamazole and other related substances
Carry out the test protected from light and prepare the solutions immediately before use.
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
ı(1) Disperse a quantity of the powdered tablets containing 20 mg of Carbimazole in 10 ml of
acetonitrile with the aid of ultrasound for 5 minutes, filter through a nylon syringe filter and
dilute 1 volume to 4 volumes with 5% v/v of acetonitrile.
ı(2) 0.00025% w/v of carbimazole BPCRS in 5% v/v of acetonitrile.
ı(3) 0.0005% w/v of thiamazole in 5% v/v of acetonitrile.
ı(4) 0.01% w/v of carbimazole BPCRS and 0.0005% w/v of thiamazole in 5% v/v of
acetonitrile.
CHROMATOGRAPHIC CONDITIONS
ı(a) Use a stainless steel column (15 cm × 3.9 mm) packed with base-deactivated
octadecylsilyl silica gel for chromatography (5 μm) (Waters Symmetry C18 is suitable).
ı(b) Use linear gradient elution using the mobile phases described below.
ı(c) Use a flow rate of 1 ml per minute.
ı(d) Use ambient column temperature.
ı(e) Use a detection wavelength of 254 nm.
ı(f) Inject 20 μl of each solution.
MOBILE PHASE
Mobile phase Aıacetonitrile (5% v/v)
Mobile phase Bıacetonitrile (20% v/v)

> is this nylon syringe filter equivalent with the one im using?

> i have posted the method from the BP 2009.

this is the original method, which im trying to use before

[bisnettrj2]

I think PVDF should be fine with acetonitrile, and the fact that you're not seeing the same peak in a filtered blank pretty much rules out the syringe filters.

[jackie1016]

hi again

i got the right chromatogram!

i have tried to run my sample and my sst (solution that have the carbimazole and thiamazole std) in Agilent HPLC using the BP method. i havent altered any of the procedure.

and finally this is what ive got for my sample..



this one is my SST:



both do not have the unknown peak.. then i tried running the same sample (same vial) using the same procedure and column in waters HPLC but this is what ive got...




i dont know why when i run the sample using waters hplc the unknown peak appears, is there a problem with the detector?

and also the response is different, in agilent the area of thiamazole peak is bigger than the carbimazole peak but in waters the carbimazole peak is much bigger than the thiamazole, the retention times are all the same..


do you have any clue?



thank you!

[Peter Skelton]

What wash solutions are you using on the Waters system? Typically Waters systems inject a portion of the wash solution when they make an injection, so you could be inadvertantly injecting your unknown. Worth making fresh wash solution to see, and make sure that the wash system is purged thoroughly.

The peak areas will vary between different detectors, this is just down to the optics and the accounting that the different systems use. This means that you can't compare the raw data from the two systems directly, you have to process to get comparable numbers.

[jackie1016]

oh i see thanks for the information.,

i just use 20% methanol, is it ok?

i also prime it the before each run, i also prime the needle wash..

should i also use 5% acetonitrile as my wash solution?

how come i have not see the unknown peak in my mobile phase, i use the same wash

is it possible?



thanks!

[Peter Skelton]

You don't generally see distinct peaks from contaminants in mobile phase when you're running isocratic or step gradient methods, the contaminant just gives a bit of baseline offset due to increased absorbance. However if you don't see a peak from injecting samples of your mobile phase then your mobile phase is probably clean. Try injecting a sample of the wash solution.

[jackie1016]

ok i will do that.

but can i use acetonitrile as my wash solution instead?

i dont know what is the correct wash solution., i just use the 20% methanol because that is what we are using before. i have not change it

thanks

[HPLCaddict]

The peak areas will vary between different detectors, this is just down to the optics and the accounting that the different systems use. This means that you can't compare the raw data from the two systems directly, you have to process to get comparable numbers.

True, but the area ratio of two peaks should be about the same even on different systems. If I understood correctly, the ratio is very different on the Agilent and the Waters:

>and also the response is different, in agilent the area of thiamazole peak is bigger than the carbimazole peak but in waters the >carbimazole peak is much bigger than the thiamazole, the retention times are all the same..

This ineed should not happen.

@jackie1016: Double-check that you're using the correct detection wavelength on both systems. As far as I can see, the Agilent has a DAD and you're using a reference wavelength? What about the Waters system?

[Peter Skelton]

Oops, indeed you should get the same ratios system to system once all's said and done.

Things to check: make sure your detector paramaters are all exactly the same. Wavelength, references, sampling rates, time constants all need to be set correctly.

Also worth checking: make sure you are using the right injection mode on the Waters system, if you only want to inject a small volume then you need to be using partial loop injections or have the correct size loop installed if you're performing full loop injections. The Agilent uses a different injector design so doesn't have this issue.

For wash solvent, I tend to use the same organic concentration as my strong solvent, so in your case 20% ACN. Try it and see if you have any issues with peak shape and if it helps with your extra peak.

[jackie1016]

hi again

i have tried running my sample again for the last time.. i've given up already..
we'll be sourcing another API supplier to confirm if the API has the problem..

but i have some fews questions though..

during this last trial, i have replaced the wash solution with 100% acetonitrile, i've cleaned and sonicated the port filters, and ive changed the line that im using for the gradient (before i was using line C and Line D for my MPA and MPB) now i have used Line A and B for the mobile phases.. before i start the run i prime the wash solvent and the needle wash.. i run 100% acetonitrle in my column (symmetry shield 150 x 4.6 ) for 15 minutes then i equilibrated the column with 5% acn ( i just run 5% acn for 30 minutes)...

(note : i have used the BP method during this run.)

this is the chromatogram for my thiamazole :

the unknown peak that i have seen before is not seen in this chromatogram now..



there is no unknown peak now...

then i have run the carbimazole API only but i still got the unknown peak.. (i have run this one without the reference wavelength.. the one that i run in the agilent has the reference wavelength at 360nm, and i think this one made the unknown peak disappear, but i cant use this reference wavelength because in this wavelength the carbimazole peak is also detected., )



also with my sst.. its all the same for carbimazole..

..

i really dont know what to do then i tried do the test for api RELATED SUBSTANCES using the bp.. with this conditions...

Column:
— size: l = 0.15 m, Ø = 3.9 mm,
— stationary phase: octadecylsilyl silica gel for chromatography R (5 µm).
i used the waters symmetry shield for my column..
Mobile phase: acetonitrile R, water R (10:90 V/V).
detection : 254 nm
injection volume :10 ul
Flow rate: 1 mL/min.

5 mg the sample (carbimazole API was dissolved and diluted in 10 ml of 20% ACN)

i have no other impurities in my carbimazole api. and i have only 1 peak...



then i tried running the same sample i used in my gradient run in isocratic condition using 20% ACN MPB..
i still get 1 peak for my carbimazole, and a thiamazole peak..



why do i have an unknown peak beside my carbimazole peak during the gradient run, but when i run the same sample in isocratic ung my MPB (20% acn) i only got i clear peak for carbimazole..

can anyone help me..

i still got time until the new batch of API arrive..

thank you so much!!

[carls]

Do the peak areas of the carbimazole match between the different conditions? If not, do they match if you add the impurity peak to the carbimazole peak? How many injections of each soltution were masde with each set of conditions; hopefully more than one and they were reproducible. If this behavior is reproducible it looks like there is an impurity in your carbimazole. You definately need to turn off the reference wavelength if your analyte absorbs there.